For simplicity, vaccinia virus (VV) genes are routinely grouped into early or late classes depending on whether their expression is independent of, or dependent on, viral DNA synthesis. Very little is known concerning the mechanisms which govern the switch between early and late gene expression modes during the VV replicative cycle. Recently a considerable amount of data has been obtained concerning the structure, nucleotide sequence, and regulation of some "typical" early VV genetic loci. This information should provide the basis for the subsequent identification of VV early gene regulatory signals and the factors which recognize them. To date however, similar analyses have not been carried on the VV late genes. It is towards this problem that the experiments outlined in this proposal are directed. The genomic location of a number of VV late genes will be determined by two methods. First, DNA-mediated marker rescue techniques will be employed to map the positions of temperature-sensitive or drug-resistant VV mutants which exhibit a defective late phenotype in vivo. Second, VV late mRNA will be translated in cell-free protein synthesizing systems and late gene products identified on the basis of their enzymatic activities (virion enzymes) or polypeptide structure (e.g., VP62, or VP11). These cell-free assays can then be coupled with hybrid-arrest or hybrid-selection procedures in order to map these functions. Once representative VV late genes have been located, state-of-the-art recombinant DNA and molecular biology techniques will be used to study the structure of the transcriptional units, how they are expressed and regulated during infection, and the nature and activity of the encoded polypeptides. When compared and contrasted to the information already available concerning VV early genes, these results should provide some insight into the mechanisms which VV employs to achieve the ordered expression of it's complex developmental program within the infected cell.